EP3700922B1 - Transmembranprotein inkorporierendes vesikel - Google Patents

Transmembranprotein inkorporierendes vesikel Download PDF

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Publication number
EP3700922B1
EP3700922B1 EP18793199.3A EP18793199A EP3700922B1 EP 3700922 B1 EP3700922 B1 EP 3700922B1 EP 18793199 A EP18793199 A EP 18793199A EP 3700922 B1 EP3700922 B1 EP 3700922B1
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poly
membrane
block
ethylene glycol
vesicles
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French (fr)
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EP3700922A1 (de
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Mariana SPULBER
Dana Cristina TVERMOES
Radoslaw GORECKI
Frederik HAUGSTED
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Aquaporin AS
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Aquaporin AS
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L71/00—Compositions of polyethers obtained by reactions forming an ether link in the main chain; Compositions of derivatives of such polymers
    • C08L71/02—Polyalkylene oxides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D63/00—Apparatus in general for separation processes using semi-permeable membranes
    • B01D63/02—Hollow fibre modules
    • B01D63/04—Hollow fibre modules comprising multiple hollow fibre assemblies
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D63/00—Apparatus in general for separation processes using semi-permeable membranes
    • B01D63/10—Spiral-wound membrane modules
    • B01D63/107—Specific properties of the central tube or the permeate channel
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D69/00—Semi-permeable membranes for separation processes or apparatus characterised by their form, structure or properties; Manufacturing processes specially adapted therefor
    • B01D69/08—Hollow fibre membranes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D69/00—Semi-permeable membranes for separation processes or apparatus characterised by their form, structure or properties; Manufacturing processes specially adapted therefor
    • B01D69/12—Composite membranes; Ultra-thin membranes
    • B01D69/1213—Laminated layers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D69/00—Semi-permeable membranes for separation processes or apparatus characterised by their form, structure or properties; Manufacturing processes specially adapted therefor
    • B01D69/12—Composite membranes; Ultra-thin membranes
    • B01D69/1214—Chemically bonded layers, e.g. cross-linking
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D69/00—Semi-permeable membranes for separation processes or apparatus characterised by their form, structure or properties; Manufacturing processes specially adapted therefor
    • B01D69/14—Dynamic membranes
    • B01D69/141—Heterogeneous membranes, e.g. containing dispersed material; Mixed matrix membranes
    • B01D69/142—Heterogeneous membranes, e.g. containing dispersed material; Mixed matrix membranes with "carriers"
    • B01D69/144—Heterogeneous membranes, e.g. containing dispersed material; Mixed matrix membranes with "carriers" containing embedded or bound biomolecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D71/00—Semi-permeable membranes for separation processes or apparatus characterised by the material; Manufacturing processes specially adapted therefor
    • B01D71/06—Organic material
    • B01D71/76—Macromolecular material not specifically provided for in a single one of groups B01D71/08 - B01D71/74
    • B01D71/80—Block polymers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/04—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring from cyclic ethers only
    • C08G65/06—Cyclic ethers having no atoms other than carbon and hydrogen outside the ring
    • C08G65/08—Saturated oxiranes
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L89/00—Compositions of proteins; Compositions of derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G2650/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G2650/28—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule characterised by the polymer type
    • C08G2650/50—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule characterised by the polymer type containing nitrogen, e.g. polyetheramines or Jeffamines(r)
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G2650/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G2650/28—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule characterised by the polymer type
    • C08G2650/58—Ethylene oxide or propylene oxide copolymers, e.g. pluronics
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L2203/00—Applications
    • C08L2203/02—Applications for biomedical use

Definitions

  • the disclosure relates to a vesicle incorporating a transmembrane protein, a method of preparing vesicles incorporating a transmembrane protein, a separation membrane comprising a vesicle incorporating a transmembrane protein, and a method of preparing a thin film composite layer immobilizing vesicles incorporating a transmembrane protein on a porous substrate membrane.
  • Polymersomes or polymeric vesicles are self-assembled structures formed by amphiphilic block copolymers in a suitable solvent (eg. water), and present an inner empty cavity surrounded by a bilayered wall that can incorporate various structures, like transmembrane proteins.
  • a suitable solvent eg. water
  • the stability of polymer vesicles is increasing with the molecular weight of the forming polymer and their permeability with increasing of hydrophilic to hydrophobic ratio.
  • Block copolymers of polyoxyethylene-polyoxypropylene-polyoxyethylene (PEO-PPO-PEO), commonly known as Pluronic, with low hydrophilic to hydrophobic ratio values can form vesicles and therefore are suitable to obtain permeable membranes. Unfortunately, they are not easy to self-assemble by direct dissolution.
  • Pluronics with small PPO/PEO ratio can self-assemble in the presence of anionic surfactant or inorganic salts (as sodium dodecyl sulfate or NaF) forming structures around 800 nm up to 3000 nm.
  • anionic surfactant or inorganic salts as sodium dodecyl sulfate or NaF
  • Pluronics with small PPO/PEO ratio can self-assemble in the presence of anionic surfactant or inorganic salts (as sodium dodecyl sulfate or NaF) forming structures around 800 nm up to 3000 nm.
  • anionic surfactant or inorganic salts as sodium dodecyl sulfate or NaF
  • Pluronics are considered non-toxic and are widely used in drug delivery systems and provide exciting opportunities for gene therapy.
  • Feng Li Pluronic polymersomes stabilized by core cross-linked polymer micelles, Soft Matter, 2009, 5, 4042-4046 .
  • Many studies have shown the potential use of Pluronics as an adjuvant to increase both cell-mediated and antibody-mediated immune responses when used with a broad spectrum of antigens ( Jain-Gupta N, et al, Pluronic P85 enhances the efficacy of outer membrane vesicles as a subunit vaccine against Brucella melitensis challenge in mice, FEMS Immunol Med Microbiol 66 (2012) 436-444 ).
  • Block copolymers including pluronics as pluronic-polylactic copolymer self-assembles in vesicles that can load and transport various cargo (eg. insulin) (X iong XY Vesicles from Pluronic/poly(lactic acid) block copolymers as new carriers for oral insulin delivery, Journal of Controlled Release 120 (2007)11-17 ).
  • cargo eg. insulin
  • X iong XY Vesicles from Pluronic/poly(lactic acid) block copolymers as new carriers for oral insulin delivery, Journal of Controlled Release 120 (2007)11-17 .
  • Pluronics can exhibit biological activity including effects on enhancing DNA cellular uptake, nuclear translocation, and gene expression.
  • the Pluronics with a higher hydrophilic-lipophilic balance value lead to homogeneous distribution in the cytoplasm; those with a lower hydrophilic-lipophilic balance value prefer to localize in the nucleus ( Fan W et al. Degradable gene delivery systems based on Pluronics-modified low-molecular-weight polyethyleneimine: preparation, characterization, intracellular trafficking, and cellular distribution, International Journal of Nanomedicine 2012:7 1127-1138 ).
  • Vesicles comprising transmembrane proteins, such as aquaporins, can be used to make membranes having immobilized Aquaporins for applications such as the purification of water ( WO2006/122566 ) or the generation of salinity power ( WO2007/033675 ).
  • the vesicles are generally deposited as a layer or in a film on a supporting substrate, which allows the selective passage of water molecules through the membranes by nanofiltration, reverse osmosis, forward osmosis or pressure retarded osmosis.
  • WO2013/043118 discloses thin film composite (TFC) membranes in which vesicles incorporating aquaporin water channels (AQPs) are immobilized. In addition, it discloses a method of producing thin film composite membranes and their uses in filtration processes, such as nanofiltration and osmotic filtration processes.
  • WO2010/146365 describes preparation of TFC-aquaporin-Z (AqpZ) filtration membranes that use an amphiphilic triblock copolymer as a vesicle forming substance for incorporating immobilized AQPs.
  • WO2014/108827 discloses a hollow fiber (HF) module having fibers modified with a thin film composite (TFC) layer comprising aquaporin water channels in which the aquaporin water channels may be incorporated in lipid or block copolymeric vesicles before incorporation into the TFC layer.
  • HF hollow fiber
  • TFC thin film composite
  • the amphiphilic lipids and block copolymers used in vesicle production are solids that need to be dissolved in harsh solvents, such as tetrachloromethane (CCl 4 ) or chloroform (CHCl 3 ), to solubilize their predominantly hydrophobic portions.
  • harsh solvents such as tetrachloromethane (CCl 4 ) or chloroform (CHCl 3 )
  • this solvent is evaporated to allow film formation which is then rehydrated to bring the amphiphile into various emulsion forms (such as vesicles), with simultaneous incorporation of the AQP membrane protein. It would be desirable to develop a process which uses solvents having a lower environmental impact.
  • Industrial membranes may be treated at elevated temperatures during the separation process or the cleaning process.
  • the membrane When the membrane is used for treatment of foodstuff such as dairy products it is generally desired to disinfect the membranes to avoid the development of microorganisms.
  • the prior art membranes having incorporated vesicles tend to deteriorate fast when exposed to elevated temperatures.
  • the present invention relates to a vesicle incorporating a transmembrane protein, wherein the vesicle membrane forming material comprises a mixture of poly(ethylene glycol)- block- poly(propylene glycol)-block-poly(ethylene glycol) and polyetheramine.
  • the vesicle according to the invention generally withstand elevated temperature without substantial shrinkage of the diameter.
  • the low shrinkage of the vesicle diameter results in a high mechanical dimensional stability of the membrane, which in turn provides for long production life. Furthermore, the low shrinkage maintains the water permeability virtually unaffected.
  • the poly(ethylene glycol)-block-poly(propylene glycol)- block -poly(ethylene glycol) usually is a substantially linear polymer having a weight average molecular weight of between about 1,000 Da to about 15,000 Da, such as a weight average molecular weight of between about 2,500 Da to about 10,000 Da.
  • the weight average molecular weight is above 3,000 Da, such as above 4,000 Da and preferably above 5,000 Da.
  • the weight average molecular weight of this aspect is not higher than 8,000 Da, such as not higher than 7,000 Da, and preferably not higher than 6,000 Da.
  • the weight average molecular weight is around 5,800 Da.
  • the poly(ethylene glycol)- block -poly(propylene glycol)- block -poly(ethylene glycol) may have different composition and proportion of the blocks but generally the compound may be represented by the chemical formula: in which
  • x denotes an integer between 15-25
  • y denotes an integer between 60-80
  • z denotes an integer between 15-25.
  • x and y have the same meaning.
  • x and z are preferably around 20 and y is around 70.
  • This compound is available as Pluronic P-123.
  • x and z are in the range of 30-200 and y is in the range 40-60.
  • the polyetheramine usually contains one or more primary amino groups attached to a polyether backbone.
  • the polyether backbone is normally based on propylene oxide (PO) or a mixture of propylene oxide (PO) and ethylene oxide (EO).
  • PO propylene oxide
  • EO ethylene oxide
  • the molar ratio PO/EO is usually higher than 1, i.e. polyetheramine is predominately polypropylene glycol (PPG) based.
  • PPG polypropylene glycol
  • the molar ratio PO/EO is usually higher than 2, such as higher than 3.
  • the polyether backbone may contain 1 to 3 amine groups, i.e. the polyether is a monoamine, diamine, or triamine. In a preferred aspect of the invention the polyether amine is a monoamine. When a mixture of propylene oxide (PO) and ethylene oxide (EO) is applied the amine group is predominantly positioned at the end of the propylene oxide (PO) part of the molecule.
  • PO propylene oxide
  • EO ethylene oxide
  • the molecular weight of the polyetheramine generally ranges from 500 to 5000 Da. In a certain aspect of the invention, the molecular weight is from 1000 to 4000 Da, such as 1500 to 3000 Da. In a preferred aspect of the invention the molecular weight of the polyetheramine is around 2000 Da.
  • the polyetheramine is of the general structure in which
  • n/m is 1 or more, such as 2 or 3 or more. More suitable, m designates an integer of 2 to 10, such as 4 - 8. Most preferred around 6. n is suitably in the range of 10 to 40, such as in the range of 25 to 35.
  • the proportion between the poly(ethylene glycol) -block- poly(propylene glycol)- block -poly(ethylene glycol) and the polyetheramine may be selected with in broad ranges. Usually, however, the proportion by weight between the poly(ethylene glycol)- block -poly(propylene glycol)- block -poly(ethylene glycol) and the polyetheramine is 5 to 1.
  • the poly(ethylene glycol)- block -poly(propylene glycol)- block -poly(ethylene glycol) forms the backbone of the vesicle, in which the propylene glycol units assemble into a hydrophobic domain and the ethylene glycol protrudes into the extravesicular space.
  • the hydrophobic part of the polyetheramine will anchor the molecule in the vesicle, resulting in the amine groups to be protruding into the extravesicular space.
  • the polyetheramine may be said to decorate the surface of the vesicles with amine groups.
  • Transmembrane proteins span in their natural environment the entire bilipid membrane, i.e. from the interior of the cell to the extracellular space. Many of the transmembrane proteins functions as gateways for specific substances, thereby allowing exchange of these substances between the interior of the cell and the extracellular liquid.
  • a characteristic feature of transmembrane proteins is the presence of a hydrophobic area, which will ensure integration of the transmembrane protein into the membrane.
  • the transmembrane protein furthermore has hydrophilic segments on both sides of the hollow fiber area, said hydrophilic segments being directed to the interior of the cell and the extracellular fluid, respectively.
  • the transmembrane protein is incorporated into the hydrophobic part of the vesicles.
  • transmembrane protein that transport ions (ion channels) and water (aquaporin water channels).
  • Ion channels include chloride channels and metal ion transporters. Chloride channels in addition to the chloride ion also conducts HCO 3 - , I - , SCN - , and NO 3 - in some transmembrane proteins.
  • the metal ion transporters include magnesium transporters, potassium ion channels, sodium ion channels, calcium channels, proton channels etc.
  • the transmembrane protein is an aquaporin water channel.
  • Aquaporin water channels facilitate the transport of water in or out of a cell.
  • the aquaporin water channels ensure the flow of water by osmosis, while others salutes in the solution are rejected.
  • Transmembrane proteins tend to aggregate and precipitate in aqueous solutions and it may therefore be suitable that the transmembrane protein is solubilized in a detergent. While a number of detergent may be used, generally the detergent is selected from the group consisting of lauryldimethylamine N-oxide (LDAO), octyl glucoside (OG), dodecyl maltoside (DDM) or combinations thereof.
  • LDAO lauryldimethylamine N-oxide
  • OG octyl glucoside
  • DDM dodecyl maltoside
  • the invention also relates to a method of preparing vesicles incorporating a transmembrane protein comprising the steps of
  • the vesicles prepared may also be referred to as polymersomes or polymeric vesicles.
  • the vesicles are self-assembled structures formed by amphiphilic block copolymers in a suitable solvent (eg. water) during the agitation step.
  • a suitable solvent eg. water
  • the vesicles present an inner empty cavity surrounded by a bilayered wall that can incorporate various structures, like transmembrane proteins.
  • the average molecular weight of the poly(ethylene glycol)- block- poly(propylene glycol)-block-poly(ethylene glycol) is at least 1000 Dalton.
  • a too high molecular weight tends to be difficult to assemble into a vesicle.
  • the average molecular weight of the poly(ethylene glycol)- block- poly(propylene glycol)- block -poly(ethylene glycol) is preferably not above 15,000 Dalton.
  • the poly(ethylene glycol)- block- poly(propylene glycol)- block -poly(ethylene glycol) is a substantially linear polymer having a weight average molecular weight of between about 2,500 Da to about 10,000 Da.
  • the poly(ethylene glycol)- block -poly(propylene glycol)- block- poly(ethylene glycol) has a molecular weight of 10,000 to 15,000 Da.
  • the permeability of the vesicle generally increases with increasing hydrophilic to hydrophobic ratio.
  • the amount of propylene glycol units is generally higher than the amount of ethylene glycol units.
  • Block copolymers of polyoxyethylene-polyoxypropylene-polyoxyethylene (PEO-PPO-PEO), commonly known as Pluronic have low hydrophilic to hydrophobic ratio values and therefor are suitable to obtain permeable membranes.
  • the poly(ethylene glycol)-block-poly(propylene glycol)- block- poly(ethylene glycol) has the chemical formula: in which
  • the polyetheramine is suitably of the general structure in which
  • the proportion by weight between the poly(ethylene glycol)- block -poly(propylene glycol)- block -poly(ethylene glycol) and the polyetheramine is 5 to 1.
  • the transmembrane protein Before the aqueous transmembrane protein, such as aquaporin, is mixed with the polyetheramine in step a, suitably the transmembrane protein is solubilized in a detergent.
  • the solubilization of the transmembrane protein in a detergent prevent or ameliorate the tendency of the transmembrane to precipitate in the aqueous solution.
  • the detergent is selected from the group consisting of lauryldimethylamine N-oxide (LDAO), octyl glucoside (OG), dodecyl maltoside (DDM) or combinations thereof.
  • the vesicles produced as disclosed above is included in a separation membrane.
  • the separation membrane comprises an active layer incorporating the vesicle and a porous support membrane.
  • the porous support membrane should not substantially impede the flux of water and/or the ion transported by the transmembrane protein.
  • the main purpose of the porous support membrane is to serve as a scaffold for the active layer incorporating the vesicles, thus allowing the transmembrane protein to be the predominate discriminating element.
  • the active layer comprises the vesicle incorporated in a thin film composite layer formed on a porous substrate membrane.
  • the vesicles containing amine groups on the surface will be not only physically incorporated or immobilized in (adsorbed), but, in addition, chemically bound in the TFC layer, because the reactive amine groups, will participate in the interfacial polymerization reaction with the acyl chloride, such as a trimesoyl chloride (TMC).
  • TMC trimesoyl chloride
  • vesicles will be covalently bound in the TFC layer, leading to relatively higher vesicle loading and thus higher water flux through the membranes.
  • the covalent coupling of vesicles in the TFC layer results in higher stability and/or longevity of the aquaporins and aquaporin-incorporated vesicles when incorporated in the selective membrane layer.
  • transmembrane protein comprises an ion channel or an aquaporin or the like
  • said vesicles comprising said transmembrane protein are immobilized or incorporated in said active or selective layer
  • separation membranes or filtration membranes having diverse selectivity and transport properties, e.g. ion exchange membranes when said transmembrane protein is an ion channel, or water filtration membranes when said transmembrane protein is an aquaporin.
  • the transmembrane protein maintains its biologically active folded structure when complexed into the self-assembled vesicles wherein it may be shielded from degradation. Even sensitive amphiphilic proteins may become sufficiently stable and, thus, preserve their desired functionality when processed into separation membranes in lab and industrial scale.
  • the present invention further relates to a method of preparing a thin film composite layer immobilizing vesicles incorporating a transmembrane protein on a porous substrate membrane, comprising the steps of
  • the di-amine compound may be selected among a range of compounds including for example, phenylenediamines, such as m-phenylenediamine, p-phenylenediamine, 2,5-dichloro-p-phenylenediamine, 2,5-dibromo-p-phenylenediamine, 2,4,6-trichloro-m-phenylenediamine, 2,4,6-tribromo-m-phenylenediamine, etc; diaminobiphenyls, such as 2,2'-diaminobiphenyl, 4,4'-diaminobiphenyl, 3,3'-dichloro-4,4'-diaminobiphenyl, 3,5,3',5'-tetrabromo-4,4'-diaminobiphenyl, etc; diaminodiphenylmethanes, such as 4,4'-diaminodiphenylmethane, 3,3'-diaminodiphen
  • N-phenyl-benzene-1,3 diamine melanine, and mixtures of such compounds.
  • diamine is selected as m-phenylenediamine (MPD) also known as 1,3-diaminobenzene.
  • the tri-amine compound may be selected among a range of compounds including for example, diethylene triamine, dipropylene triamine, phenylenetriamine, bis(hexamethylene)-triamine, bis(hexamethylene)triamine, bis(3-aminopropyl)-amine, hexamethylenediamine, N-tallowalkyl dipropylene, 1,3,5-triazine-2,4,6-triamine, and mixtures of these compounds.
  • the acyl halide compound usually has two or three acyl halide groups available for reaction with the di- or triamine compound.
  • Suitable examples of diacyl halide or triacyl halide compounds include trimesoyl chloride (TMC), trimesoyl bromide, isophthaloyl chloride (IPC), isophthaloyl bromide, terephthaloyl chloride (TPC), terephthaloyl bromide, adipoyl chloride, cyanuric chloride and mixtures of these compounds.
  • the amine groups of the di-amine or tri-amine compound will compete with the acid chloride groups of the acyl halide compound for reaction.
  • the proportion by weight of the di-amine or tri-amine compound to acyl halide compound is from 0:1 to 30:1.
  • the amount of di-amine or tri-amine groups is usually in the lower part of the range, i.e. 0:1 to 1:1, such as between 0:1 to 0.5:1.
  • a more rigid TFC layer is desired and a selection of the reactants are in the higher end of the range, such as 1:1 to 30:1, preferably 1:1 to 5:1.
  • the aqueous amine solution may be added to the porous support membrane in an even layer and subsequently dried before the application of the acyl halide solution.
  • the aqueous amine solution is applied to the porous support membrane and subsequently a vacuum is provided on the opposing side of the porous support membrane to stimulate the permeation of the aqueous amine solution into the porous structure.
  • the acyl chloride solution is applied for the formation of the thin film composite layer by the reaction of the amine with the acyl chloride.
  • the use of a vacuum is believed to provide for a better integration of the thin film composite layer in the porous support membrane.
  • the porous support membrane may be formed by a number of materials.
  • the specific choice of material is not essential as long as the support membrane is able sufficiently to support the TFC layer and to withstand decomposition during operation condition, i.e. able to withstand the pressure and/or the chemical environment on either side of the membrane.
  • Specific examples of materials for the porous support membrane include polysulfone or a polyethersulfone polymer.
  • the support may be symmetrical or asymmetrical. In the case the porous support membrane is asymmetrical, the TFC layer is suitably formed on the skin layer face.
  • the porous support membrane may further be supported by a woven or non-woven mechanical support in some embodiments to increase the mechanical construction and reduce the risk of fractures during operation.
  • the porous support membrane may any physical appearance known in the art, such as flat sheet membrane, tubular membrane, or hollow fiber membrane.
  • a hollow fiber membrane is preferred as it provides for higher packing density, i.e. the active membrane area is higher for a certain volume.
  • the membranes may be grouped together or assembled into a module as known in the art.
  • a plurality of flat sheet membranes may be assembled into a plate-and-frame membrane configuration.
  • Plate-and-frame membrane systems utilize membranes laid on top of a plate-like structure, which in turn is held together by a frame-like support.
  • Flat sheet membranes may also be assembled into spiral-wound filter modules.
  • the spiral-wound membrane modules include feed spacers, and permeate spacers wrapped around a hollow tube called the permeate tube.
  • Spiral wound elements utilize cross flow technology, and because of its construction, can easily be created in different configurations with varying length, diameter, and membrane material.
  • a spiral-wound filter module may be produced by first laying out a membrane and then fold it in half with the membrane facing inward. Feed spacer is then put in between the folded membranes, forming a membrane sandwich. The purpose of the feed spacer is to provide space for water to flow between the membrane surfaces, and to allow for uniform flow between the membrane leaves.
  • the permeate spacer is attached to the permeate tube, and the membrane sandwich prepared earlier is attached to the permeate spacer using glue.
  • the next permeate layer is laid down and sealed with glue, and the whole process is repeated until all of the required permeate spacers have been attached to the membranes.
  • the finished membrane layers then are wrapped around the tube creating the spiral shape.
  • Tubular membrane modules are tube-like structures with porous walls. Tubular modules work through tangential cross-flow and are generally used to process difficult feed streams such as those with high dissolved solids, high suspended solids, and/or oil, grease, or fats. Tubular modules consist of a minimum of two tubes; the inner tube, called the membrane tube, and the outer tube, which is the shell. The feed stream goes across the length of the membrane tube and is filtered out into the outer shell while concentrate collects at the opposite end of the membrane tube.
  • the hollow fiber membranes may be assembled into a module.
  • the present invention provides the step of producing a hollow fiber module by assembling a bundle of hollow fibers in a housing, wherein an inlet for passing a first solution is connected to the lumen of the hollow fibers in one end and an outlet is connected to the lumen in the other end, and an inlet is provided in the housing for passing a second solution to an outlet connected to the housing.
  • the membrane modules produced in accordance with the present invention may be used in various configurations, including forward osmosis configurations and reverse osmosis configurations.
  • the present invention also relates to vesicles comprising an internal cargo, wherein the charged vesicles are capable of releasing the internal cargo upon pH change in the environment.
  • the vesicles comprising a mixture of poly(ethylene glycol)-block-poly(propylene glycol)- block- poly(ethylene glycol) and polyetheramine are stable at stable at alkaline pH values, such as below pH 7 or pH 8 or above.
  • acid values such as pH values of pH 7 or below or pH 6 or below
  • the vesicle formation dissociates.
  • the pH selective behavior of the vesicles offers the opportunity for incorporating cargo in the vesicles at alkaline pH values, transporting the vesicles with cargo to a desired location and releasing the cargo by subjecting the vesicles to acid conditions, which will allow the vesicles to dissociate.
  • the vesicles for cargo delivery have the same composition and preparation method as described above for vesicles incorporation transmembrane proteins. Due to the preparation method, the vesicles are very at alkaline pH values, i.e. pH from pH 7 to pH 14 and temperature from 30 to 90°C. Initial tests suggest that the vesicles are stable for at least one year at room temperature without any change.
  • the vesicles reassemble at neutral or basic pHs making easy the incorporation of internal cargo when needed without any additional purification steps.
  • the vesicles comprising a cargo substance may be used as a carrier system for the in situ or in vivo delivery of various cargo substances, including bioactive moieties.
  • the cargo substance may be a bioactive substance, for example, such as a bioactive substance selected from the group consisting of small molecule drugs, biomolecules, biomacromolecules and cells.
  • the bioactive substance may be supported on a non-bioactive carrier.
  • the cargo substance may be a polymeric or inorganic particle.
  • Illustrative examples of substances that may be utilized as cargo substances in accordance with the present invention include, but are limited to, the following : small molecule drugs, biomolecules, biomacromolecules (including, but not limited to, polysaccharides, glycosaminoglycans, and proteins), cells (including live cells), therapeutic agents (i.e., agents that cause a measurable physiological response in an animal, such as a human), fluorophores, chromagenic agents, enzymes, proteins (including immunomodulatory proteins and matrix metalloproteinases), antibiotics, anesthetics, antibodies, growth factors, hormones, anti-inflammatories, analgesics, cardiac agents, psychotropics, fillers (e.g., inorganic and/or polymeric particles), immunotherapeutics, cytokines, oligonucleotides, labels (e.g., fluorophores, radionucleotides, fluorescent moieties, chemiluminescent moieties, magnetic particles, dyes) and
  • the present invention relates to the use of a mixture of poly(ethylene glycol)-block-poly(propylene glycol)- block- poly(ethylene glycol) and polyetheramine, to form self-assembled vesicles with transmembrane proteins, such as aquaporin water channels.
  • the vesicles having incorporated the transmembrane protein may then be used in the production of separation membranes in which the transmembrane proteins are incorporated or immobilized, for example for allowing water molecules to pass through the membrane.
  • the vesicles may be added to an aqueous liquid composition comprising an aromatic amine, such as a diamine or triamine, e.g.
  • 1,3-diaminobenzene applied to the surface of a porous support structure, which when brought into contact with a solution of an acid chloride in an organic solvent will participate in an interfacial polymerization reaction to form a thin film composite active or selective layer on said support thus forming a separation membrane, wherein said vesicles have become immobilized or incorporated.
  • the vesicles containing free available NH 2 reactive groups on the surface will be not only physically incorporated or immobilized in (adsorbed), but, in addition, chemically bound in the TFC layer, because the NH 2 reactive groups, will participate in the interfacial polymerization reaction with the acyl chloride, such as a trimesoyl chloride (TMC).
  • TMC trimesoyl chloride
  • transmembrane protein comprises an ion channel or an aquaporin or the like
  • said vesicles comprising said transmembrane protein are immobilized or incorporated in said active or selective layer
  • novel separation membranes or filtration membranes having diverse selectivity and transport properties, e.g. ion exchange membranes when said transmembrane protein is an ion channel, or water filtration membranes when said transmembrane protein is an aquaporin.
  • transmembrane protein maintains its biologically active folded structure when complexed into the self-assembled nanostructures wherein it may be shielded from degradation, even sensitive amphiphilic proteins may become sufficiently stable and, thus, preserve their desired functionality when processed into separation membranes in lab and industrial scale.
  • the separation membrane of the invention is useful in an industrial or domestic setting for preparing a pure water filtrate, such as filtering an aqueous solution through a separation membrane in a nanofiltration process or in a reverse osmosis process.
  • separation membrane includes selectively permeable membranes and semipermeable membranes for water filtration and water separation, such as asymmetric membranes comprising a porous support membrane having a selective layer formed on one side, such as a thin crosslinked aromatic polyamide layer or film or a layer of alternately charged polyelectrolytes (L-B-L).
  • the other side may be reinforced by a woven or non-woven layer or mesh typically made of polyester fibers.
  • the separation membrane of the invention is useful in a method for the concentration of a product solution, said method comprising utilizing a separation membrane of the invention mounted in a filter housing or module to extract water from the product solution, e.g. by forward osmosis.
  • the selective layer comprises a thin film composite (TFC) layer formed on the inside surface of the fibers through an interfacial polymerization reaction, wherein said TFC layer comprises aquaporin water channels incorporated in vesicles composed of a mixture of poly(ethylene glycol)-block-poly(propylene glycol)- block- poly(ethylene glycol) and polyetheramine.
  • TFC thin film composite
  • the separation membrane of the invention may additionally be useful in a method for the production of salinity power using pressure retarded osmosis, said method comprising utilizing said separation membrane to increase hydrostatic pressure, and using the increase in hydrostatic pressure as a source of salinity power, cf. WO2007/033675 and WO2014128293 (A1 ).
  • aquaporin water channel includes a functional natural or synthetic aquaporin or aquaglyceroporin water channel, such as aquaporin Z (AqpZ), GlPf, SoPIP2;1, aquaporin 1 and/or aquaporin 2.
  • Aquaporin water channels include bacterial aquaporins and eukaryotic aquaporins, such as yeast aquaporins, plant aquaporins and mammalian aquaporins, as well as related channel proteins, such as aquaglyceroporins.
  • aquaporins and aquaglyceroporins include: prokaryotic aquaporins such as AqpZ; mammalian aquaporins, such as Aqp1 and Aqp2; plant aquaporins, such as plasma intrinsic proteins (PIP), tonoplast intrinsic proteins (TIP), nodulin intrinsic proteins (NIP) and small intrinsic proteins (SIP), e.g. SoPIP2;1, PttPIP2;5 and PtPIP2;2; yeast aquaporins, such as AQY1 and AQY2; and aquaglyceroporins, such as GlpF and Yfl054.
  • prokaryotic aquaporins such as AqpZ
  • mammalian aquaporins such as Aqp1 and Aqp2
  • plant aquaporins such as plasma intrinsic proteins (PIP), tonoplast intrinsic proteins (TIP), nodulin intrinsic proteins (NIP) and small intrinsic proteins (SIP), e
  • Aquaporin water channel proteins may be prepared according to the methods described herein or as set out in Karlsson et al. (FEBS Letters 537: 68-72, 2003 ) or as described in Jensen et al. US 2012/0080377 A1 (e.g. see Example 6).
  • separation membrane includes membranes useful for separating water and, optionally, certain small size solutes including anions and cations, from other solutes, particles, colloids and macromolecules.
  • separation membranes are "filtration membranes” such as nanofiltration (NF) membranes, forward osmosis (FO) membranes and reverse osmosis (RO) membranes.
  • filtration membranes such as nanofiltration (NF) membranes, forward osmosis (FO) membranes and reverse osmosis (RO) membranes.
  • NF nanofiltration
  • FO forward osmosis
  • RO reverse osmosis
  • One type of filtration membranes is a "thin film composite” (or TFC) membrane, often classified as nanofiltration and reverse osmosis membranes.
  • Flat sheet TFC membranes are typically made by depositing a polyamide layer on top of a polyethersulfone or polysulfone porous layer on top of a non-woven or woven fabric support.
  • the polyamide rejection layer is formed through interfacial polymerization of an aqueous solution of an amine with a solution of an acid chloride in an organic solvent.
  • TFC membranes may be produced as described in WO 2013/043118 (Nanyang Technological University & Aquaporin A/S).
  • Other types of filtration membranes are those formed by the layer-by-layer (LbL) deposition method, such as described in Gribova et al. (Chem. Mater., 24: 854-869, 2012 ) and Wang et al. (Membranes, 5(3): 369-384, 2015 ) .
  • the vesicles of the invention may be embedded or incorporated in the polyelectrolyte multilayer (PEM) films, as outlined in Figure 4 of Gribova et al.
  • PEM polyelectrolyte multilayer
  • Thin-film-composite or (TFC) membranes as used herein may be prepared using an amine reactant, preferably an aromatic amine, such as a diamine or triamine, e.g.,1,3-diaminobenzene (m-Phenylenediamine, > 99%, e.g. as purchased from Sigma-Aldrich) in an aqueous solution, and an acyl halide reactant, such as a di- or triacid chloride, preferably an aromatic acyl halide, e.g. benzene-1,3,5-tricarbonyl chloride ( CAS No. 84270-84-8 , trimesoyl chloride (TMC), 98%, e.g.
  • an aromatic amine such as a diamine or triamine, e.g.,1,3-diaminobenzene (m-Phenylenediamine, > 99%, e.g. as purchased from Sigma-Aldrich
  • Benzene-1,3,5-tricarbonyl chloride (trimesoyl chloride) is dissolved in a solvent, such as a C 6 -C 12 hydrocarbon including hexane (>99.9%, Fisher Chemicals), heptane, octane, nonane, decane etc. (straight chain or branched hydrocarbons) or other low aromatic hydrocarbon solvent, e.g. Isopar TM G Fluid which is produced from petroleum-based raw materials treated with hydrogen in the presence of a catalyst to produce a low odour fluid, the major components of which include isoalkanes.
  • a solvent such as a C 6 -C 12 hydrocarbon including hexane (>99.9%, Fisher Chemicals), heptane, octane, nonane, decane etc. (straight chain or branched hydrocarbons) or other low aromatic hydrocarbon solvent, e.g. Isopar TM G Fluid which is produced from petroleum-based raw
  • Isopar TM G Fluid Chemical Name: Hydrocarbons, C10-C12, isoalkanes, ⁇ 2% aromatics; CAS No: 64742-48-9, chemical name: Naphtha (petroleum), hydrotreated heavy (from ExxonMobil Chemical).
  • Alternatives to the reactant 1,3-diaminobenzene include diamines such as hexamethylenediamine etc., and alternatives to the reactant benzene-1,3,5-tricarbonoyl chloride include a adipoyl chloride, cyanuric acid etc. as known in the art.
  • the vesicles of the present invention may be referred to "self-assembled" to describe the process by which vesicles are formed through hydrophilic and hydrophobic interaction of the amphiphilic substances, i.e. the mixture of poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) and polyetheramine.
  • Hydrodynamic diameter represents the hydrodynamic size of nanoparticles in aqueous media measured by dynamic light scattering (DLS) defined as the size of a hypothetical hard sphere that diffuses in the same fashion as that of the particle being measured.
  • DLS dynamic light scattering
  • Forward osmosis or direct osmosis is an osmotic process that uses a selective and permeable membrane to effect separation of water from dissolved solutes.
  • the driving force for this separation is an osmotic pressure gradient between a solution of high concentration, herein referred to as the draw and a solution of lower concentration, referred to as the feed.
  • the osmotic pressure gradient induces a net flow of water through the membrane into the draw, thus effectively concentrating the feed.
  • the draw solution can consist of a single or multiple simple salts or can be a substance specifically tailored for forward osmosis applications.
  • the feed solution can be a dilute product stream, such as a beverage, a waste stream or seawater, cf. IFOA, http://forwardosmosis.biz/education/what-is-forward osmosis/.
  • PAFO pressure assisted forward osmosis process
  • PRO pressure retarded osmosis which is useful in the generation of osmotic power.
  • Membranes of the present invention are useful in all types of forward osmosis processes and may be specifically adapted for each forward osmosis type.
  • RO reverse osmosis
  • Reverse osmosis refers to when an applied feed water pressure on a selectively permeable membrane is used to overcome osmotic pressure. Reverse osmosis typically removes many types of dissolved and suspended substances from feed water, including bacteria, and is used in both industrial processes and in the production of potable water. During the RO process, the solute is retained on the pressurized side of the membrane and the pure solvent, the permeate, passes to the other side. Selectivity specifies that the membrane does not allow larger molecules or ions through its pores (holes), while allowing smaller components of the solution (such as solvent molecules) to pass freely.
  • LPRO membranes typically operates at a feed water pressure of from about ⁇ 5 bar and up to a maximum operating pressure of about 25 bar 15 specific flux LMH/bar. LPRO performed at the lower feed pressure ranges, e.g. 2 to 5 bar is sometimes designated ultra-low pressure reverse osmosis. LPRO membranes known in the art have typical operating limits for feed water temperature of about 45 °C, feed water pH in the range of 2 to 11, and chemical cleaning in the range of pH 1 to 12.
  • Pluronic ® P-123 triblock copolymer poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) having a composition of PEG20-PPO70-PEG20 with molecular weight of 5800 Da was purchased from Sigma Aldrich and was used as received.
  • Jeffamine ® M-2005 is a polyetheramine with the ratio polyethylene oxide polypropylene oxide of 29 to 6 and molecular weight of 2000 Da and was purchased from Huntsman and was used as received.
  • Phosphate buffer 10 mM (PBS) (pH 7.2, 136 mM NaCl, 2.6 mM KCl) was prepared by dissolving 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4 and 0.24 g of KH2PO4 in 800 mL MiliQ purified H2O, adjusting the pH to 7.2 with HCL and completing the volume to 1 L.
  • Aquaporin Z 5 mg/mL stock solution was prepared as disclosed below. Functional aquaporin-Z was overproduced in E. coli strain BL21(DE3) bacterial cultures as His-tagged protein with a tobacco etch virus cleavage site. The fusion protein has 264 amino acid and a Mw of 27234 Da. Genomic DNA from E. coli DH5 was used as a source for amplifying the AqpZ gene. The AqpZ gene was amplified using gene specific primers with the addition of a tobacco etch virus cleavage site (TEV); ENLYFQSN at the N-terminus of AqpZ.
  • TSV tobacco etch virus cleavage site
  • the amplified AqpZ was digested with the enzyme NdeI and BamHI and then ligated to the similarly digested 6-His tagged expression pET28b vector DNA.
  • the positive clones were verified by PCR-screening. The authenticity of the constructs was then confirmed by DNA sequencing.
  • the E. coli strain BL21(DE3) was used for expression of the protein.
  • Luria Broth cultures containing 50 ⁇ g/ml kanamycin were incubated for 13-16 hours at 37C, diluted 100-fold into fresh LB broth and propagated to a density of about 1.2-1.5 (OD at 600 nm).
  • Expression of recombinant protein was induced by addition of 1 mM IPTG for 3 hour at 35°C before centrifugation.
  • the flow though fraction was topped up with NaCl to 300 mM before loaded onto a pre-equilibrated Ni-NTA column.
  • the column was washed with 100 column volumes of a wash buffer (20 mM Tris pH 8.0, 300 mM NaCl, 25 mM imidazole, 2 mM ⁇ -mercaptoethanol, 10% glycerol) to remove non-specifically bound material.
  • Ni-NTA agarose bound material was eluted with five bed volumes of elution buffer (20 mM Tris pH 8.0, 300 mM NaCl, 300 mM imidazole, 2 mM ⁇ -mercaptoethanol, 10% 15 glycerol, containing 30 mM n-octyl ⁇ -D-Glucopyranoside).
  • AqpZ was further purified with anion exchange chromatography; monoQ column (GE healthcare). The sample mixture was diluted and concentrated to bring the salt and imidazole concentration to approximately 10 mM with Amicon concentrator, membrane cut off 10,000 Da before loading to MonoQ column.
  • the buffer used during anion exchange chromatography were (A) 20 mM Tris pH 8.0, 30 mM OG, 10% glycerol and (B) 20 mM 20 Tris pH 8.0, 1 M NaCl, 30 mM OG, 10% glycerol.
  • the eluted peak fractions containing AqpZ from the ion exchange column was pooled.
  • the purified AqpZ extract was kept frozen at -80°C.
  • the AQP extract (stored at -80 °C freezer) was thawed on ice or in a 4°C refrigerator. Portions of the buffers and ddH2O were readied at 4°C.
  • the AQP extract was stirred in an adequate chilled beaker on ice bath by a magnetic stick to dissolve any precipitate.
  • 1.5 volumes of pre-chilled LDAO-free AQP binding buffer was gradually added into 1 volume of the solubilized extract (using a further 0.5 volume buffer for rinsing the extract tubes and filtration cup), mixed well and filtered through a sterile 0.45 ⁇ M vacuum filter cup. Vacuum was applied to the filter cup to avoid excess foaming and the filtrate was placed on ice to use within 2 hours.
  • a Histrap column was equilibrated with sterile water followed by AQP Binding buffer at RT.
  • the flow rate was set at 1ml/min (for 1 mL prepacked column) or 2.5 ml/min (for 5 ml prepacked column and self-packed column).
  • the 3 times diluted extract (on ice water bath) was loaded onto the Histrap column using ⁇ KTA program.
  • the flow rate was set at 1 ml/min (for 1 mL prepacked column) or 2.5 ml/min (for 5 mL prepacked column and self-packed column).
  • the loading volume was less than 30 ml/ml resin.
  • the extract flow-through on ice-water bath was collected and stored at 4°C for further use.
  • the column was washed with 10 CV (column volume) ice cold AQP binding buffer.
  • the flow rate was set at 2.5 ml/min (for 5 ml prepacked column and self-packed column) or set at 1 ml/min for 1 ml prepacked column.
  • the AQP protein was eluted with ice cold AQP elution buffer (10 column volume) at flow rate 2.5 ml/min using ⁇ KTA program.
  • the fraction volume was set to 10 ml and collection started in 15 mL PP tubes after 0.5 - 1CV.
  • Eluted fractions were capped and stored on ice or 4 °C.
  • the AQP purity and conformation was examined by denaturing and native PAGE analysis respectively. Protein concentration was measured by Nanodrop.
  • the extract flow-through may be processed a second and a third time as needed to produce an AQP composition of suitable quality.
  • the protein concentration was adjusted to 5 mg/ml by adding ice cold imidazole-free AQP binding buffer containing 2% LDAO. Finally, the AQP was sterilized by filtration through 0.45 ⁇ M sterilized cup and stored at 4°C in refrigerator for use within a month or else stored at -80°C in a freezer.
  • Temperature stability and thermal behavior were tested by warming up 5 mL of vesicle formulation for 10 min at various temperatures ranging from 30°C to 100°C and their size and water permeability was further determined by DLS and stopped-flow measurements.
  • Thermal treatment does not affect significantly the stability of the formulation, resulting in the diameter shrinkage of the larger size structures from around 200 nm at room temperature to around 1800 nm. From water permeability point of view no changes can be observed up to 100°C. Ki values from 1700 to 1687 s-1 were recorded.
  • the pH behavior shows the disassemble of vesicle formulation at pH varying from 1 to 7 to micelles with a diameter up to 20 nm and reassembling at basic pH values (from 9 to 13 showing the same size 180 nm and Ki values around 1700 s-1.
  • the membrane After removal of the tape the membrane can be transferred to MilliQ with the newly formed active side up and keep wet during handling in subsequent steps if necessary.
  • TFC membranes with liquid AQPZ formulation of 5.5 cm x 11 cm sizes was then be mounted in a Sterlitech CF042 FO cell (www.sterlitech.com) and subjected to tests of 60 minutes (5 membranes) and tests of 900 minutes (4 membranes) duration in FO mode using deionised (MilliQ) water as feed and 1 M NaCl aqueous solution as draw and feed and draw speeds of 268 mL/min.
  • Sterlitech CF042 FO cell www.sterlitech.com
  • Vesicles incorporating AqpZ were prepared by firstly mixing the aqueous solution of transmembrane protein (Aquaporin Z stock solution as prepared above) with polyetheramine (15 mg/mL Jeffamine ® M-2005) to obtain a final concentration of 1/200 AQPZ/polymer molar protein ratio. Subsequently, adding PEO-PPO-PEO aqueous solution (Pluronic ® P-123 having a molecular weight of 5800 Da in PBS to a final concentration of 10 mg/mL), and agitating the mixture overnight at 170 rotations per min at room temperature.
  • PEO-PPO-PEO aqueous solution Pluronic ® P-123 having a molecular weight of 5800 Da in PBS to a final concentration of 10 mg/mL
  • Such prepared vesicles were incorporated into the polyamide thin-film composite (TFC) membrane, by interfacial polymerization on the porous support.
  • Aqueous solution was prepared comprising the vesicle mixture (6 ml of the mixture prepared above) and m-phenylenediamine solution (prepared by dissolving 1.5 g MPD in 52.5 ml MilliQ).
  • the organic solution comprised of trimesoyl chloride (TMC) and Isopar TM E in a concentration of 0.15% W/V.
  • the coating protocol comprised soaking the porous support with aqueous solution, followed by gentle removal of its excess. Subsequently, organic solution was applied and polyamide layer was formed, excess of organic solution was gently dried. The membranes were stored in miliQ water prior testing.
  • Vesicle properties Ki 1412s -1 , pH 9.83, Zeta potential -0.339 (average), size: 204nm (average), 100% population.
  • the dimensions of the extruded vesicles were determined by dynamic light scattering using ZetaSizer NanoZs from Malvern.
  • the water flux through AQP channels was tested using a Bio-Logic SFM 300 stopped-flow device, using a monochromator at 517 nm and a cut off filter at 530 nm.
  • the porous support was mounted in a suction cell with the active layer facing upwards, and a vacuum pump applied underneath, facing the inactive layer.
  • the support for used for the TFC layer was MicroPES 1F PH microporous support from Membrana GmbH. 50 mL of aqueous solution containing MPD and Formulation 10-2-10 in RO water was poured into the suction cell, covering the porous support. Afterwards, a suction of 100 mBar was applied for 5 minutes, sucking the MPD and formulation onto the support. Vacuum was turned off and 50 mL of organic solution containing TMC and Isopar-E was applied and given 1 minute of reaction time to facilitate the interfacial polymerization. The organic solution was then flushed out, and the membrane was left to dry for 3 minutes and was then transferred to a petri dish with RO water until ready for QC testing.
  • Formulation 10-2-10 10 mg/ml Pluronic f127 (poloxamer 407) - 2 mg/ml Jeffamine M2005 - 10 mg/ml aquaporin stock solution. in PBS buffer (137 mM NaCl, 2,7 mM KCl, 10 mM Na 2 HPO 4 and 2 mM of KH 2 PO 4 ).
  • the vesicle formulations were both tested for size, water permeability and zeta potential point of view by DLS, Zeta potential and stopped flow measurements in 0.5 M NaCl.
  • the dimensions of the vesicles are determined by dynamic light scattering using ZetaSizer Nano ZS from Malvern.
  • the water flux through vesicle membrane is tested using a Bio-Logic SFM 300 stopped-flow (SF) device, using a monochromator at 517 nm and a cut-off filter at 530 nm.
  • SF stopped-flow
  • Table 8 shows the osmotic coefficient k i , which is calculated based the exponential growth of the stopped-flow light scattering results for the vesicles incorporating Aquaporin Z and blank ones. The analysis of exponential growth is made on the first population of the structures showing the most rapid shrinkage.
  • the osmotic coefficient k i (s -1 ) is directly proportional with the water flux through polymeric membrane and the results show that the presence of aquaporins in the vesicles significantly increases the water flux through polymeric membrane.
  • the other properties of the vesicles are substantially unaffected by the presence of aquaporin Z, i.e. the hydrodynamic diameter, zeta potential and the pH remain at the same level.
  • TFC FO membranes with Aquaporin Z formulation of 5.5 cm x 11 cm sizes were then mounted in a Sterlitech CF042 FO cell (www.sterlitech.com) and subjected to tests of 200 minutes duration in FO mode, using 5 ⁇ M calcein in deionised (MilliQ) water as feed and 1 M NaCl aqueous solution as draw and feed and draw speeds of 50 mL/min.
  • Sterlitech CF042 FO cell www.sterlitech.com
  • Table 9 shows the results of FO experiment with membranes incorporating vesicles incorporating Aquaporin Z proteins and comparison to the blank ones (control membranes). It may be concluded that Jv increases by the incorporation of the vesicles incorporating Aquaporin Z and that the Js/Jv remains at the same level.

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Claims (15)

  1. Vesikel, das ein Transmembranprotein enthält, wobei das vesikelbildende Material eine Mischung aus Poly(ethylenglycol)-Block-Poly(propylenglycol)-Block-Poly(ethylenglycol) und Polyetheramin umfasst.
  2. Vesikel nach Anspruch 1, wobei es sich bei dem Poly(ethylenglycol)-Block-Poly(propylenglycol)-Block-Poly(ethylenglycol) um ein im Wesentlichen lineares Polymer mit einer durchschnittlichen Molekülmasse zwischen etwa 1.000 Da bis etwa 15.000 Da handelt.
  3. Vesikel nach einem der Ansprüche 1 bis 2, wobei das Poly(ethylenglycol)-Block-Poly(propylenglycol)-Block-Poly(ethylenglycol) die folgende chemische Formel hat:
    Figure imgb0009
     in der
    x eine ganze Zahl zwischen 10 und 30 bezeichnet
    y eine ganze Zahl zwischen 50 und 100 bezeichnet
    z eine ganze Zahl zwischen 10 und 30 bezeichnet.
  4. Vesikel nach einem der Ansprüche 1 bis 3, wobei das Polyetheramin die folgende allgemeine Struktur hat
    Figure imgb0010
     in der
    m eine ganze Zahl von 1 bis 15 ist
    n eine ganze Zahl von 5 bis 50 ist
    R = CH3 ist.
  5. Vesikel nach einem der Ansprüche 1 bis 4, wobei es sich bei dem Transmembranprotein um einen Aquaporin-Wasserkanal handelt.
  6. Verfahren zum Herstellen von Vesikeln, die ein Transmembranprotein enthalten, umfassend die Schritte
    a. Mischen einer wässrigen Lösung von Transmembranprotein und Polyetheramin,
    b. Zugeben von Poly(ethylenglycol)-Block-Poly(propylenglycol)-Block-Poly(ethylenglycol) als wässrige Lösung zu der in Schritt a hergestellten Mischung,
    c. Rühren der in Schritt b erhaltenen Lösung.
  7. Verfahren nach Anspruch 6, wobei es sich bei dem Poly(ethylenglycol)-Block-Poly(propylenglycol)-Block-Poly(ethylenglycol) um ein im Wesentlichen lineares Polymer mit einer durchschnittlichen Molekülmasse zwischen etwa 1.000 Da bis etwa 15.000 Da handelt.
  8. Verfahren nach den Ansprüchen 6 oder 7, wobei das Poly(ethylenglycol)-Block-Poly(propylenglycol)-Block-Poly(ethylenglycol) die folgende chemische Formel hat:
    Figure imgb0011
     in der
    x eine ganze Zahl zwischen 10 und 30 bezeichnet
    y eine ganze Zahl zwischen 50 und 100 bezeichnet
    z eine ganze Zahl zwischen 10 und 30 bezeichnet.
  9. Verfahren nach einem der Ansprüche 6 bis 8, wobei das Polyetheramin die folgende allgemeine Struktur hat
    Figure imgb0012
     in der
    m eine ganze Zahl von 1 bis 15 ist
    n eine ganze Zahl von 5 bis 50 ist
    R = CH3 ist.
  10. Verfahren nach einem der Ansprüche 6 bis 9, wobei es sich bei dem Transmembranprotein um einen Aquaporin-Wasserkanal handelt.
  11. Trennmembran, umfassend ein Vesikel nach einem der Ansprüche 1 bis 5.
  12. Trennmembran nach Anspruch 11, wobei die Trennmembran eine aktive Schicht umfasst, die das Vesikel und eine poröse Trägermembran enthält.
  13. Verfahren zum Herstellen einer Dünnfilm-Verbundschicht, die Vesikel immobilisiert, die ein Transmembranprotein auf einer porösen Substratmembran enthalten, umfassend die Schritte:
    a. Bereitstellen einer wässrigen Lösung umfassend die gemäß einem der Ansprüche 6 bis 10 hergestellten Vesikel und eine Diamin- oder Triaminverbindung,
    b. Bedecken der Fläche einer porösen Trägermembran mit der wässrigen Lösung von Schritt a,
    c. Auftragen einer hydrophoben Lösung umfassend eine Acylhalogenidverbindung und
    d. Ablaufenlassen einer Grenzflächenpolymerisationsreaktion der wässrigen Lösung und der hydrophoben Lösung, um die Dünnfilm-Verbundschicht zu bilden.
  14. Verfahren nach Anspruch 13, wobei es sich bei der porösen Trägermembran um eine Hohlfaser handelt.
  15. Verfahren nach den Ansprüchen 13 oder 14, wobei es sich bei der porösen Trägermembran um ein flaches Blatt handelt.
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EP3800223A1 (de) * 2019-10-04 2021-04-07 Sika Technology Ag Reinigungszusammensetzung für reaktive klebstoffe und verwendung davon
CN112023727B (zh) * 2020-08-14 2022-07-12 万华化学集团股份有限公司 一种抗污染抑菌反渗透膜、制备方法及其应用
CN112892230B (zh) * 2021-02-01 2022-04-26 浙江工业大学 一种用于海水淡化的高脱盐聚酰胺复合反渗透膜及其制备方法

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US20030031911A1 (en) * 2001-04-13 2003-02-13 Rosalyn Ritts Biocompatible membranes and fuel cells produced therewith
ATE457823T1 (de) 2005-05-20 2010-03-15 Aquaporin Aps Membran zum filtrieren von wasser
ES2377234T3 (es) 2005-09-20 2012-03-23 Aquaporin A/S Membrana de agua biomimética que comprende acuaporinas usadas en la producción de energía de sanilidad
MA33094B1 (fr) 2009-02-03 2012-03-01 Aquaz As Nanofabrication d'une membrane au moyen de protéoliposomes polymérisés
DK177144B1 (en) 2009-06-19 2012-02-06 Aquaporin As A liquid membrane suitable for water extraction
DK177307B1 (en) 2010-12-17 2012-11-12 Aquaporin As A liquid membrane
SG11201400825XA (en) * 2011-09-21 2014-04-28 Univ Nanyang Tech Aquaporin based thin film composite membranes
US10226744B2 (en) 2012-10-19 2019-03-12 Danisco Us Inc Stabilization of biomimetic membranes
GB201300465D0 (en) 2013-01-11 2013-02-27 Aquaporin As A hollow fiber module having tfc-aquaporin modified membranes
GB201405390D0 (en) 2014-03-26 2014-05-07 Applied Biomimetic As Process for making membranes
DK3347031T3 (da) 2015-09-11 2021-04-26 Nutrition & Biosciences Usa 1 Llc Sammensætning, der omfatter et protein og en polyalkoxyfedtforbindelse

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CN111417649A (zh) 2020-07-14
JP7326303B2 (ja) 2023-08-15
SG11202002669SA (en) 2020-05-28
IL273898A (en) 2020-05-31
DK3700922T3 (da) 2022-03-07
EP3700922A1 (de) 2020-09-02
IL273898B (en) 2022-11-01
KR20200077545A (ko) 2020-06-30
JP2021501053A (ja) 2021-01-14
IL273898B2 (en) 2023-03-01
US20200239685A1 (en) 2020-07-30
CN111417649B (zh) 2024-04-26

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